Electrostatic Swelling and Conformational Variation Observed in High

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Electrostatic Swelling and Conformational Variation Observed in High-Generation Polyelectrolyte Dendrimers )

Yun Liu,†,‡ Chun-Yu Chen,§ Hsin-Lung Chen,*,§ Kunlun Hong, Chwen-Yang Shew,^ Xin Li,#,3 Li Liu,3 Yuri B. Melnichenko,# Gregory S. Smith,# Kenneth W. Herwig,# Lionel Porcar,*,O and Wei-Ren Chen*,#,[,z †

The NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-6100, Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, §Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, The Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, ^Department of Chemistry, City University of New York, College of Staten Island, Staten Island, New York 10314, #Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, 3Department of Mechanical, Aerospace & Nuclear Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, OInstitut Laue-Langevin, B.P. 156, F-38042 Grenoble CEDEX 9, France, [Department of Chemical & Biomolecular Engineering, The University of Tennessee, Knoxville, Tennessee 37996-2200, and zJoint Institute for Neutron Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 )

‡

ABSTRACT A combined small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) study was conducted to investigate the structural characteristics of aqueous (D2O) solution of generation 7 and 8 (G7 & G8) polyamidoamine (PAMAM) dendrimer as a function of molecular protonation. A consequent change in the intramolecular dendrimer conformation was clearly quantified by a detailed data analysis separating intermolecular correlations from the intramolecular contribution. Our results unambiguously reveal both an increase in the molecular size and a continuous variation of the intramolecular density profile upon increasing molecular protonation. This observation is contrary to current understanding of high-generation polyelectrolyte dendrimers where steric crowding is supposed to stiffen the local motion of dendrimer segments hindering exploration of available intradendrimer free volume and thereby inhibiting electrostatic swelling. Our observation is relevant to the elucidation of the general microscopic picture of polyelectrolyte dendrimer structure, as well as the development of dendrimer-based packages based on the stimuli-responsive principle. SECTION Macromolecules, Soft Matter

olyamidoamine (PAMAM) dendrimers are man-made spherical-like polyelectrolyte macromolecules with a sophisticated hyperbranched organization. Their structural study is challenging from a theoretical standpoint because of the complexity arising from a single molecule having both polymeric and colloidal characteristics.1 Moreover, when dissolved in acidified aqueous environments, the binding between free protons and the constituent amines of PAMAM dendrimer provides a molecular protonation tunable by simply adjusting the pH of the solution. The influence of this additional electrostatic interaction and the prospect of using this charge-stimulated conformational evolution to facilitate and enhance dendrimer applications as hosts for encapsulation of molecules for targeted drug carriers, gene therapy scaffolds, and water decontaminating agents have provided motivation for extensive studies.1,2 Complemented by molecular dynamics (MD) simulation3 and theoretical analysis within a framework provided by statistical mechanics, small-angle scattering techniques, including neutron (SANS) and X-ray (SAXS), prove to be an effective

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r 2010 American Chemical Society

means to explore the structural evolution of polyelectrolyte dendrimers.4 A quantitative SANS study of fourth-generation (G4) of PAMAM dendrimer dissolved in D2O solutions first revealed that, upon increasing the molecular protonation, the internal structural conformation evolved from a densecore configuration for the neutral state to a more uniform, outstretched distribution when fully charged. However, only a minor increase of the overall molecular size (less than 4% increase in the radius of gyration RG) was observed,5 in contradiction to the significant molecular swelling predicted by early computational studies.3 With an improved force field, recent atomistic MD simulations carried out by Goddard and co-workers confirmed this minor increase in radius of gyration.6 They have attributed this modest expansion to enhanced counterion association, which was pointed out previously by

Received Date: May 12, 2010 Accepted Date: June 9, 2010 Published on Web Date: June 15, 2010

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DOI: 10.1021/jz1006143 |J. Phys. Chem. Lett. 2010, 1, 2020–2024

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Figure 1. The SANS absolute intensity I(Q) obtained from of G7 (a) and G8 (b) PAMAM dendrimer solutions at three different R values and the corresponding model fitting (black solid lines). For the sake of clarity, I(Q) is scaled by the indicated factor. The shift of the high order correlation peaks is readily discerned in the insets.

Crooks and co-workers.7 The latest MD simulation carried out by Kzos and Sommer8 calculated the scattering functions corresponding to a dendrimer under moderate size variation at different levels of molecular protonation, and the results are in quantitative agreement with our previous SANS measurements.5 Although a recent computational work continues to predict a significant molecular expansion upon charging,9 a modest electrostatic swelling of low and medium generation (G3-6) polyelectrolyte dendrimers has been repeatedly observed by a majority of the quantitative experimental structural studies10-12 and predicted by computational investigations having improved computational algorithms and potentials.6,8,13-15 While a consistent picture of modest electrostatic swelling has developed for G3 to G6 dendrimers, the structural responsiveness of higher generation dendrimers is less clear. An earlier SANS study conducted by Amis and co-workers pointed out the disappearance of charge-induced expansion of G8 PAMAM dendrimer dissolved in D2O.16 In their experimental spectra, the position of the form factor local minimum remained invariant with changing pH. Using the indirect Fourier transform (IFT) analysis that approximately models the single G8 dendrimer as a homogeneous hard sphere, the radius of gyration of the dendrimer RG was observed to remain constant upon charging. This conformational independence with molecular protonation has been conjecturally attributed to the effect of steric crowding, which increasingly hinders any backfolding motion of dendrimer segments with each advancing generation.1 This reported size invariance is in contradiction with MD results17 that predicted considerable electrostatic swelling for G8 dendrimers. However, this prediction was based on a force field in which the strength and range of the interaction among the constituent components of the simulated system were not optimized as specifically pointed out in a more recent study by the same group.6 It is also important to note that application of this improved force field has not been made to higher generation dendrimers, but did produce results consistent with measurement for low to medium generation dendrimers.


To resolve this controversial issue, we have carried out SANS experiments at the Oak Ridge National Laboratory HFIR18 and the National Institute of Standards and Technology Center for Neutron Research19 on D2O solutions of G7 and G8 dendrimers prepared following procedures described elsewhere.5,10-12 In this study, DCl was used to charge the amines of PAMAM dendrimers. The acidity of the samples is given by R, the molar ratio of DCl to that of the terminal amines. The neutron wavelength was set at 7.0 ((0.8) Å with neutron guide selected to enhance the resolution at the relevant range of scattering wave-vector Q. A previously developed mean-field model to account for the partially nonuniform dendrimer interior due to segmental flexibility was employed in the SANS data analysis to quantify the pH (pD) responsiveness of the dendrimer structure.5 Figure 1 gives the SANS absolute intensity I(Q) obtained from a solution of G7 (Figure 1a) and G8 (Figure 1b) PAMAM dendrimer in D2O at a concentration of 0.0225 g/mL and at three different R values. Similar to the behavior observed in lower generation PAMAM dendrimers, progressive protonation generates a local ordering suggested by the presence and enhancement of a correlation peak (centering at ∼0.04 Å-1 for G7 and ∼0.03 Å-1 for G8). Unlike the scattering intensities presented in ref 16, which were insensitive to the structural charge of a dendrimer, the high-Q correlation peak that contains information on the intramolecular structure is clearly seen to shift toward lower Q with increasing protonation. This difference arises from a combination of effects including a poorer instrumental resolution employed in the previous study, which limited the ability of the investigators to observe the shift seen in this work, as well as an intrinsic limitation in their choice of a hard sphere model to analyze the data. Structural flexibility and the ability to redistribute mass from the central to outer regions of the dendrimer is a central feature of our model along with consideration of interdendrimer interactions that are non-negligible in the charged state. As indicated in the Supporting Information (SI), this qualitative observation conclusively demonstrates a

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Figure 2. (a) Radius of gyration (RG, open symbols) of PAMAM dendrimer dissolved in D2O as a function of dendrimer generation at three different levels of molecular protonation. (b) Parameter Δ, which is defined as the difference between the values of RG obtained at R = 1.6 and R = 0 as a function of dendrimer generation. (c) The ratio of Δ to RG obtained at R = 0 as a function of dendrimer generation.

Figure 3. The normalized radial density distribution F(r) as a function of the radial distance r obtained at three R values for G7 (a) and G8 (b) PAMAM dendrimer solutions. The insets give the R dependence of the total mass fraction within the molecular central region with radius of 15 Å.

function of generation. Apart from G3, which exhibits a huge uncertainty due to constraints of neutron flux and instrument resolution resulting in poorer data quality, molecular expansion steadily increases in successive dendrimer generations. The percentage of swelling, which is defined as the ratio of Δ to RG obtained at R = 0 for each generation is also seen to increase steadily, as shown in Figure 2c. To visualize the details of this conformational change, the normalized radial intramolecular density distribution functions F(r), calculated from the form factor parameters R and σ, are presented in Figure 3 as a function of R. Upon increasing R from 0 to 1.6, a significant intramolecular structural rearrangement is clearly observed, as indicated by a 40% decrease in the mass fraction within the molecular central region (within a radius of 15 Å) as a result of molecular swelling. This charge-induced outward mass migration has also been reported in our previous SANS studies of lower PAMAM generations (G3-G6) in D2O solutions.

noticeable molecular swelling of dendrimer upon charging due to the electrostatic interaction among the amines. The quantitative molecular characteristics extracted from the SANS spectra of Figure 1, along with those of the lower generation reported elsewhere,5,10-12 are given in Figure 2. Several interesting features are noticed: First, when dissolved in D2O, the global size of PAMAM dendrimer quantified by RG (calculated from the effective hard-core radius R and the parameter σ used to quantify the fuzziness of molecular periphery in our soft ball form factor)5 is seen to increase linearly (blue circles) with generation, while the dendrimer molecular weight doubles in each successive generation. Second, judging from the evolution of RG with R, electrostatic swelling increases as a function of generation within experimental errors. This trend is clearly revealed in Figure 2b, which displays the dependence of Δ, the difference between the values of RG obtained at R = 1.6 and at the neutral state, as a


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high-generation of PAMAM dendrimers induced by molecular protonation. The associated intramolecular structural change observed for G7 and G8 PAMAM dendrimers upon charging seem to be a generic feature for PAMAM dendrimers from low (G3) to high generation (G8). Our observation strongly supports the existence of segmental backfolding mechanism in high generation dendrimers even in the presence of strong steric hindrance. These findings should generate valuable input and additional challenges for the theoretical and computational study of dendrimer science.

SUPPORTING INFORMATION AVAILABLE An example of SANS model fitting, the complementary SAXS experiment, the charge dependence of the intramolecular density profiles, and a disclaimer. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 4. The SANS coherent scattering cross sections calculated from the spheroid of radius 40 Å with fixed volume but different aspect ratio ε ranging from 1 (spherical objects) to 1.5.

Explicit MD simulations have provided the microscopic interpretation of this general observation as a consequence of the spatial relocation of the dendrimer terminal groups due to the interplay among positively charged amines, oppositely charged condensed counterions, and penetrating water molecules.6,8,13-15 A critical factor for segmental backfolding is to induce a sufficient intramolecular and surface spacing to accommodate rearrangement of the terminal groups. On the basis of the previous experimental measurement of G8 dendrimer,17 it has been generally believed that electrostatic swelling for polyelectrolyte dendrimers of higher generation was inhibited1 due to progressively enhanced steric crowding originating from the collectively linear growth in the dendrimer size and exponential amplification of the terminal groups as a function of generation.20-23 However, our experimental observation provides conclusive evidence of a non-negligible variation of the dendrimer radius of gyration RG with protonation. Furthermore, the change in the internal configurations seem to be a general feature characterizing the structural response of PAMAM dendrimer to a pH variation, from the flexible, open structure of lower generation to the more densely packed higher generation. In all generations, there is a redistribution of mass from the central region to the outer portions of the dendrimer with increasing protonation. Moreover, in addition to electrostatic swelling upon charging, another change in PAMAM dendrimer conformation, in terms of aspect ratio of the molecular shape, has been predicted computationally.17 It is therefore worth exploring the influence of this predicted conformational change on the measured SANS I(Q). As an example, a series of calculated coherent scattering spectra based on spheroids24 with the same volume of a sphere with radius of 40 Å is presented in Figure 4 (including instrumental resolution effects typical of our measurements). Given a fixed molecular volume, it is clear that the change in the molecular aspect ratio ε, which is defined as the ratio of primary to secondary axes, mainly results in a shift of the steep local maxima and minima toward higher Q, opposite to the behavior observed experimentally with increasing protonation. This calculation clearly demonstrates that the observed shift of the high order correlation peak in I(Q) is indeed a result of molecular swelling and not from a change in molecular shape. In summary, we provide the first compelling experimental evidence demonstrating significant increase in the size of


AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: chenw@ ornl.gov (W.-R.C.); [email protected] (L.P.); [email protected] (H.-L.C.).

ACKNOWLEDGMENT We gratefully acknowledge the support of NCNR NIST in providing the neutron research facilities. The research at Oak Ridge National Laboratory's High Flux Isotope Reactor was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Sample preparation was conducted at CNMS, which is sponsored at ORNL by the Division of Scientific User Facilities, U.S. Department of Energy. C.Y.S. acknowledges the support from the CUNY PSC grants. We also acknowledge the SAXS beam time provided by NSRRC.

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Electrostatic Swelling and Conformational Variation Observed in High

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